G?s is critical for the actions of PTH and PTHrP. The gene encoding G?s (GNAS) also gives rise to XL?s, which is expressed in various tissues including osteoblastic and renal cells. XL?s can mimic G?s by stimulating cAMP generation in response PTH, although it is also predicted to have unique actions. Mutations in GNAS are found in several human diseases that impair signaling through the PTH/PTHrP receptor (PTHR). Most of these mutations affect both G?s and XL?s. Studies in mice and humans indicate that XL?s plays important roles in physiology and human disease, but the actions of XL?s remain poorly understood. Our recent studies have provided novel insights into the cellular actions of XL?s, and together with our findings obtained from XL?s knockout (XLKO) mice, these led us to hypothesize that XL?s is necessary for the regulation of calcium and phosphorus metabolism in vivo. In Aim 1 of the current proposal, we will address whether XL?s is necessary for PTH-mediated actions in the renal proximal tubule during early postnatal development. We will determine a) whether proximal tubular actions of PTH in XLKO mice are impaired and b) whether the PTH resistance phenotype in XLKO mice is rescued by transgenically reconstituting XL?s expression in the proximal tubule. In Aim 2, we will address whether XL?s allows PTH actions to be sustained in the renal proximal tubule, which could explain the PTH resistance phenotype in XLKO mice. We will thus determine a) whether PTHR internalization is enhanced in the proximal tubule of XLKO mice; b) whether the PTH resistance phenotype in XLKO mice can be rescued by a mutant PTHR with sustained activity or by overexpressing G?s in the proximal tubule; and c) whether the interaction of XL?s with dynamin influences PTH actions. These studies will provide novel insights into the actions of PTH and XL?s in the renal proximal tubule, and these will be applicable to the actions of these proteins in skeletal tissues. Our results will also help reveal the roles of XL?s in other systems, in addition to improving our knowledge of the mechanisms underlying the diseases caused by GNAS mutations. Given that XL?s can activate the ubiquitous cAMP signaling pathway, our results will likely have even broader implications for human health and disease.